Strain Of Streptococcus Suis Serotype Research Articles

Proteomic analysis of bEnd.3 cells infected with wild-type and stk-deficient strains of Streptococcus suis serotype 2 reveals protein and pathway regulation

Streptococcus suis serotype 2 (SS2) is a zoonotic pathogen causing meningitis in humans and pigs. However, information on the comparative protein expression of the blood-brain barrier (BBB) following SS2 infection is limited. Deletion of the serine/threonine kinase (stk) gene can decrease the ability of SS2 to invade the BBB. In the present study, bEnd.3 cells were used as the BBB model, and a SILAC comparative quantitative proteomic study of bEnd.3 cells infected with the SS2 ZY05719 or Δstk strain was performed to determine the differences between these strains infections. Compared with ZY05719-infected cells, 241 proteins were highly upregulated, and 81 were significantly downregulated in Δstk-infected cells. The obtained data revealed major changes in the proteins involved in RNA process, host cytoskeleton, tight junction disruption and immune response. Some differentially expressed proteins were screened by quantitative real-time PCR to examine their regulation at the transcriptional level, and western blot analysis was used to validate the changes of some selected proteins at the translational level. The results obtained in this study may be useful to understand the host response to SS2 infection and provide crucial clues to decipher how STK expression in SS2 helps the bacteria penetrate the BBB. SignificanceA SILAC comparative quantitative proteomic assay was performed in bEnd.3 cells infected with the SS2 ZY05719 or Δstk strain. 241 upregulated and 81 downregulated differentially expressed proteins (DEPs) were identified. DEPs are involved in RNA process, host cytoskeleton, tight junction disruption and immune response. Some DEPs were examined by qPCR and western blot assays, which were similar to those of their corresponding proteins in the quantitative proteomics analysis.

Streptococcus suis Uptakes Carbohydrate Source from Host Glycoproteins by N-glycans Degradation System for Optimal Survival and Full Virulence during Infection.

Infection with the epidemic virulent strain of Streptococcus suis serotype 2 (SS2) can cause septicemia in swine and humans, leading to pneumonia, meningitis and even cytokine storm of Streptococcal toxic shock-like syndrome. Despite some progress concerning the contribution of bacterial adhesion, biofilm, toxicity and stress response to the SS2 systemic infection, the precise mechanism underlying bacterial survival and growth within the host bloodstream remains elusive. Here, we reported the SS2 virulent strains with a more than 20 kb endoSS-related insertion region that showed significantly higher proliferative ability in swine serum than low-virulent strains. Further study identified a complete N-glycans degradation system encoded within this insertion region, and found that both GH92 and EndoSS contribute to bacterial virulence, but that only DndoSS was required for optimal growth of SS2 in host serum. The supplement of hydrolyzed high-mannose-containing glycoprotein by GH92 and EndoSS could completely restore the growth deficiency of endoSS deletion mutant in swine serum. EndoSS only hydrolyzed a part of the model glycoprotein RNase B with high-mannose N-linked glycoforms into a low molecular weight form, and the solo activity of GH92 could not show any changes comparing with the blank control in SDS-PAGE gel. However, complete hydrolyzation was observed under the co-incubation of EndoSS and GH92, suggesting GH92 may degrade the high-mannose arms of N-glycans to generate a substrate for EndoSS. In summary, these findings provide compelling evidences that EndoSS-related N-glycans degradation system may enable SS2 to adapt to host serum-specific availability of carbon sources from glycoforms, and be required for optimal colonization and full virulence during systemic infection.

Isolation and characterization of a native avirulent strain of Streptococcus suis serotype 2: a perspective for vaccine development.

Streptococcus suis, an emerging infectious pathogen, is the cause of two large-scale outbreaks of human streptococcal toxic shock syndrome in China, and has attracted much attention from the scientific community. The genetic basis of its pathogenesis remains enigmatic, and no effective prevention measures have been established. To better understand the virulence differentiation of S. suis and develop a promising vaccine, we isolated and sequenced a native avirulent S. suis strain (05HAS68). Animal experiments revealed that 05HAS68 is an avirulent strain and could protect piglets from the attack of virulent strains. Comparative genomics analyses demonstrated the genetic basis for the lack of virulence in 05HAS68, which is characterized by the absence of some important virulence-associated factors and the intact 89K pathogenicity island. Lack of virulence was also illustrated by reduced survival of 05HAS68 compared to a virulent strain in pig whole blood. Further investigations revealed a large-scale genomic rearrangement in 05HAS68, which was proposed to be mediated by transposase genes and/or prophages. This genomic rearrangement may have caused the genomic diversity of S. suis, and resulted in biological discrepancies between 05HAS68 and highly virulent S. suis strains.

Suicin 3908, a New Lantibiotic Produced by a Strain of Streptococcus suis Serotype 2 Isolated from a Healthy Carrier Pig

While Streptococcus suis serotype 2 is known to cause severe infections in pigs, it can also be isolated from the tonsils of healthy animals that do not develop infections. We hypothesized that S. suis strains in healthy carrier pigs may have the ability to produce bacteriocins, which may contribute to preventing infections by pathogenic S. suis strains. Two of ten S. suis serotype 2 strains isolated from healthy carrier pigs exhibited antibacterial activity against pathogenic S. suis isolates. The bacteriocin produced by S. suis 3908 was purified to homogeneity using a three-step procedure: ammonium sulfate precipitation, cationic exchange HPLC, and reversed-phase HPLC. The bacteriocin, called suicin 3908, had a low molecular mass; was resistant to heat, pH, and protease treatments; and possessed membrane permeabilization activity. Additive effects were obtained when suicin 3908 was used in combination with penicillin G or amoxicillin. The amino acid sequence of suicin 3908 suggested that it is lantibiotic-related and made it possible to identify a bacteriocin locus in the genome of S. suis D12. The putative gene cluster involved in suicin production by S. suis 3908 was amplified by PCR, and the sequence analysis revealed the presence of nine open reading frames (ORFs), including the structural gene and those required for the modification of amino acids, export, regulation, and immunity. Suicin 3908, which is encoded by the suiA gene, exhibited approximately 50% identity with bovicin HJ50 (Streptococcus bovis), thermophilin 1277 (Streptococcus thermophilus), and macedovicin (Streptococcus macedonicus). Given that S. suis 3908 cannot cause infections in animal models, that it is susceptible to conventional antibiotics, and that it produces a bacteriocin with antibacterial activity against all pathogenic S. suis strains tested, it could potentially be used to prevent infections and to reduce antibiotic use by the swine industry.

Functional definition of LuxS, an autoinducer-2 (AI-2) synthase and its role in full virulence of Streptococcus suis serotype 2

Quorum sensing is a widespread chemical communication in response to fluctuation of bacterial population density, and has been implicated into bacterial biofilm formation and regulation of expression of virulence factors. The luxS gene product, S-ribosylhomocysteinase, catalizes the last committed step in biosynthetic pathway of autoinducer 2 (AI-2), a signaling molecule for inter-species quorum sensing. We found a luxS homologue in 05ZYH33, an epidemic strain of Streptococcus suis serotype 2 (SS2) in China. A luxS null mutant (ΔluxS) of 05ZYH33 strain was obtained using an approach of homologous recombination. LuxS was determined to be required for AI-2 production in 05ZYH33 strain of S. suis 2. Inactivation of luxS gene led to a wide range of phenotypic changes including thinner capsular walls, increased tolerance to H(2)O(2), reduced adherence capacity to epithelial cells, etc. In particular, loss of LuxS impaired dramatically its full virulence of SS2 in experimental model of piglets, and functional complementation restored it nearly to the level of parent strain. Genome-wide transcriptome analyses suggested that some known virulence factors such as CPS are down-regulated in the ΔluxS mutant, which might in part explain virulence attenuation by luxS deletion. Similarly, 29 of 71 genes with different expression level were proposed to be targets candidate regulated by LuxS/AI-2-dependent quorum sensing.

Differentiation of highly virulent strains of Streptococcus suis serotype 2 according to glutamate dehydrogenase electrophoretic and sequence type.

The glutamate dehydrogenase (GDH) enzymes of 19 Streptococcus suis serotype 2 strains, consisting of 18 swine isolates and 1 human clinical isolate from a geographically varied collection, were analyzed by activity staining on a nondenaturing gel. All seven (100%) of the highly virulent strains tested produced an electrophoretic type (ET) distinct from those of moderately virulent and nonvirulent strains. By PCR and nucleotide sequence determination, the gdh genes of the 19 strains and of 2 highly virulent strains involved in recent Chinese outbreaks yielded a 1,820-bp fragment containing an open reading frame of 1,344 nucleotides, which encodes a protein of 448 amino acid residues with a calculated molecular mass of approximately 49 kDa. The nucleotide sequences contained base pair differences, but most were silent. Cluster analysis of the deduced amino acid sequences separated the isolates into three groups. Group I (ETI) consisted of the seven highly virulent isolates and the two Chinese outbreak strains, containing Ala(299)-to-Ser, Glu(305)-to-Lys, and Glu(330)-to-Lys amino acid substitutions compared with groups II and III (ETII). Groups II and III consisted of moderately virulent and nonvirulent strains, which are separated from each other by Tyr(72)-to-Asp and Thr(296)-to-Ala substitutions. Gene exchange studies resulted in the change of ETI to ETII and vice versa. A spectrophotometric activity assay for GDH did not show significant differences between the groups. These results suggest that the GDH ETs and sequence types may serve as useful markers in predicting the pathogenic behavior of strains of this serotype and that the molecular basis for the observed differences in the ETs was amino acid substitutions and not deletion, insertion, or processing uniqueness.

Identification and distribution of putative virulent genes in strains of Streptococcus suis serotype 2

In order to identify gene sequences unique to the virulent strains, suppression subtractive hybridization (SSH) was conducted using virulent Streptococcus suis type 2 (SS2) strain HA9801 and avirulent S. suis type 2 strain T15. Thirty genomic regions were absent in T15, and the DNA sequences of these regions in HA9801 were determined. These DNA fragments, containing putative virulence genes, encoded 28 proteins that were homologous to proteins involved in various aspects of cellular surface structure, molecular synthesis, energy metabolism, regulation, transport systems and others of unknown function. According to the published SS2 genomic sequence of the Chinese strain 98HAH33, PCR primers for 14 significant DNA fragments were designed and used for detection of the distribution of these fragments in S. suis strains from different sources, serotypes, regions, groups and times. The results showed that these 14 DNA fragments were widely distributed in 37 detected SS2 strains, yet were absent among the avirulent strain T15. Moreover, these fragments could be detected in other serotypes of S. suis, but each serotype had a different distribution of the fragments.

Characterisation of biofilm formation by a Streptococcus suis meningitis isolate

Biofilm formation by a strain of Streptococcus suis serotype 2 isolated from a case of meningitis in pigs was characterised. Using a polystyrene microtitre plate assay, S. suis 95-8242 produced a dense biofilm when glucose, fructose or sucrose was used as the carbohydrate source, whereas no biofilm formed in the presence of lactose. Polysaccharide production by the biofilm-forming strain was demonstrated by the Congo red agar assay. Transmission electron microscopy revealed that bacterial cells were surrounded by a thick layer of polycationic ferritin-labelled material. S. suis 95-8242 was more resistant to both penicillin G and ampicillin in biofilms than in planktonic cultures on the basis of minimal inhibitory and minimal bactericidal concentrations.

Elimination of strains of Streptococcus suis serotype 2 from the tonsils of carrier sows by combined medication and vaccination

The effect of vaccination with a killed whole-cell vaccine of extracellular factor-positive Streptococcus suis serotype 2 (S suis 2 ef+) combined with medication with amoxicillin on the presence of virulent...

Identification of an inducible bacteriophage in a virulent strain of Streptococcus suis serotype 2.

Streptococcus suis infection is considered to be a major problem in the swine industry worldwide. Most virulent Canadian isolates of S. suis serotype 2 do not produce the known virulence markers for this pathogen. PCR-based subtraction hybridization was adapted to isolate unique DNA sequences which were specific to virulent strains of S. suis isolated in Canada. Analysis of some subtracted DNA clones revealed significant homology with bacteriophages of gram-positive bacteria. An inducible phage (named Ss1) was observed in S. suis following the incubation of the virulent strain 89-999 with mitomycin C. Phage Ss1 has a long noncontractile tail and a small isometric nucleocapsid and is a member of the Siphoviridae family. Ss1 phage DNA appears to be present in most Canadian S. suis strains tested in this study, which were isolated from diseased pigs or had proven virulence in mouse or pig models. To our knowledge, this is the first report of the isolation of a phage in S. suis.

Role of suilysin in pathogenesis of Streptococcus suis capsular serotype 2

Three suilysin (SLY) knockout mutant strains of Streptococcus suis serotype 2 were generated by allelic replacement from one North American and two European wild type strains. The mutants were characterized by Southern blot, Western blot and phenotyping. In vitro bactericidal testing showed that both wild type and SLY mutants were resistant to bactericidal factors in whole pig blood. To demonstrate the role of SLY during S. suis infection, four animal trials were carried out using young pigs. Either high dose (4×10 6 CFU/ml/pig) or low dose (0.5×10 6 CFU/ml/pig) live cell aerosol was applied to the pharynx. In one trial, a low challenge dose of North American strain SX332 and its isogenic sly − mutant strain (SX932) resulted in acute disease in 3/5 of pigs exposed to the wild type strain, while 5/5 of pigs exposed to the mutant strain survived the trial. In the repeat trial, 1/8 of pigs in wild type group and 6/8 of pigs in mutant group developed disease. The high dose trial with 332/932 pair showed that 4/8 pigs challenged with wild type and 5/8 of pigs challenged with mutant strain developed disease respectively. The third low dose trial, using European strain 31533 and its isogenic sly − mutant strain SX911, showed that 1/8 of pigs challenged with the wild type strain and 4/8 of pigs challenged with the corresponding mutant strain developed disease. All the diseased pigs showed fever, clinical signs and developed septicemia. S. suis was isolated from tissue samples such as brain, submandibular lymph node, lung, spleen, liver, heart or joint. Serum antibody titer against cell surface proteins changed little while the antibody titer against SLY increased only in the wild type group after challenge. sly gene was cloned and expressed in E. coli. The recombinant SLY (rSLY) protein showed 800 hemolysin units per μg protein. In vitro study showed that rSLY triggered TNFα production by human monocytes and IL-6 production by pig pulmonary alveolar macrophages and monocytes. Thus, the results of this study suggest that SLY does not seem to be a critical virulence factor for S. suis serotype 2 respiratory infection, but by stimulating cytokine release it may play a role in innate immunity.

Virulent strains of Streptococcus suis serotype 2 and highly virulent strains of Streptococcus suis serotype 1 can be recognized by a unique ribotype profile.

The ribotype profiles of 42 different Streptococcus suis strains were studied. These strains belonged to five serotypes and differed in their virulence for pigs as well as in the expression of the muramidase-released protein and the extracellular protein factor. For the ribotyping, chromosomal DNAs were digested with EcoRI and were hybridized with a 1,066-bp ribosomal DNA probe. The hybridization patterns showed genetic heterogeneity within and between the serotypes. Pathogenic strains of serotype 2 and highly pathogenic strains of serotype 1 could be recognized by their unique ribotype profiles. Nonpathogenic strains showed a high degree of genetic heterogeneity. Moreover, by comparing the 16S ribosomal DNA sequences of a number of S. suis strains, we were able to design two DNA probes which specifically hybridized with S. suis strains.